MicroRNA-142-3p inhibits high-glucose-induced endothelial-to-mesenchymal transition through targeting TGF-β1/Smad pathway in primary human aortic endothelial cells

Abstract

Myocardial fibrosis is an important pathological feature of diabetic cardiomyopathy (DCM) and endothelial-to-mesenchymal transition (EndMT) is an essential process for myocardial fibrosis. Recent studies have demonstrated an association between miRs and DCM. Therefore, the aim of this study is to investigate the role and the mechanism of miRNAs in the process of EndMT. We simulated the conditions occurring in EndMT by application of high glucose in primary human aortic endothelial cells (HAECs). Firstly, we compared the expression profiles of miRNAs in HAECs with or without HG treatment using microarray. Then, after addition of miR-142-3p mimics, the expression levels of EndMT markers were assessed by qRT-PCR and Western Blot. Moreover, bioinformatics analysis and luciferase assay were used to confirm the direct regulation of miR-142-3p to TGF-β1. Furthermore, the role of TGF-β1 in the inhibitory effect of miR-142-3p on EndMT was evaluated. In addition, the expressions of TGF-β1/Smad signaling signatures were measured by Western Blot. MiR-142-3p screened by miRNA microarray was significantly down-regulated in HAECs under HG stimulation in a dose and time dependent manner. Subsequently, we found that overexpression of miR-142-3p could inhibit HG-induced EndMT, as evidenced by decreased α-SMA and vimentin expression, and increased CD31 and VE-cadherin expression. Of note, transforming growth factor beta 1 (TGF-β1), one of the molecular mediators implicated in the progression of EndMT, was confirmed to be downstream target gene of miR-142-3p in HAECs. Moreover, TGF-β1 overexpression remarkably abolished the inhibitory effects of miR-142-3p overexpression on HG induced EndMT. Finally, miR-142-3p also mediated its anti-EndMT action by inactivation of TGF-β1/Smad pathway, as demonstrated by downregulation of TGF-β1, phospho-Smad2 and phospho-Smad2. Our findings demonstrated that miR-142-3p could attenuate HG-induced EndMT in HAECs, the mechanism of which may be at least partly through blocking TGF-β1/Smad signaling pathway. This might provide a potential therapeutic target for DCM in future.

Keywords: Diabetic cardiomyopathy; EndMT; TGF-β1/Smad signaling pathway; miR-142-3p.

Figure 1

miR-142-3p was involved in HG induced EndMT in HAECs. A. Morphological changes in HAECs after HG treatment. B. The protein expression of mesenchymal markers (α-SMA, FSP-1, vimentin, FN) and endothelial markers (CD31 and VE-cadherin) were determined by Western Blot. Data represent the mean ± SD of three independent experiments. **P < 0.01 vs. NC group. NC: 5 mmol/l glucose, HG: 30 mmol/l glucose. C. Heatmap of normalized expression levels of miRNAs in HAECs treated with/without HG. Green indicates low expression levels; red indicates high expression levels. D. qRT-PCR was performed to determine the expression levels of miR-142-3p in HAECs pre-treated for 5 days with 0, 10, 20, 30, or 40 mM HG. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01. E. qRT-PCR was performed to determine the expression levels of miR-142-3p in HAVECs pre-treated with 30 mM HG for 1, 2, 3, 4 or 5 days. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Figure 2

Overexpression of miR-142-3p inhibited HG-induced EndMT in HAECs. HAECs were transfected with miR-142-3p mimics and mimics NC for 24 h, and then incubated with HG for 5 days, after which the cells were harvested for subsequent experiment. A-D. The mRNA expression levels of α-SMA, vimentin, CD31 and VE-cadherin were measured by qRT-PCR. E. The protein expression levels of α-SMA, vimentin, CD31 and VE-cadherin were measured by Western Blot. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. NC group. ##P < 0.01 vs. HG group.

Figure 3

TGF-β1 was a direct target of miR-142-3p. A. The putative binding site of miR-142-3p and TGF-β1 is shown. B. Luciferase assay of HEK293 cells co-transfected with firefly luciferase constructs containing the TGF-β1 wild-type or mutated 3’-UTRs and miR-142-3p mimics, mimics NC, miR-142-3p inhibitor or inhibitor NC, as indicated (n = 3). Data represent the mean ± SD of three independent experiments. **P < 0.01 vs. mimics NC, ##P < 0.01 vs. inhibitor NC. C. The expression of TGF-β1 mRNA after transfection with miR-142-3p mimic or miR-142-3p inhibitor were measured by Western Blot. D. The expression of TGF-β1 protein after transfection with miR-142-3p mimic or miR-142-3p inhibitor were measured by Western Blot. E. The bands were semi-quantitatively analyzed by using Image J software, normalized to β-actin density. Data represent the mean ± SD of three independent experiments. **P < 0.01 vs. mimics NC, ##P < 0.01 vs. inhibitor NC.

Figure 4

miR-142-3p inhibited HG-induced EndMT by targeting TGF-β1. pcDNA-TGF-β1 and miR-142-3p mimics were co-transfected into HAECs, followed by HG treatment for 5 days, after which the cells were harvested for subsequent experiment. A-D. The mRNA expression levels of α-SMA, vimentin, CD31 and VE-cadherin were measured by qRT-PCR. E. The protein expression levels of α-SMA, vimentin, CD31 and VE-cadherin were measured by Western Blot. Data represent the mean ± SD of three independent experiments. ##P < 0.01 vs. HG + mimics group.

Figure 5

miR-142-3p improved HG-induced EndMT through TGF-β1/Smad signaling pathway. pcDNA-TGF-β1 and miR-142-3p mimics were co-transfected into HAECs, followed by HG treatment for 5 days, after which the cells were harvested for subsequent experiment. A. The protein levels of TGF-β1, Smad2, phospho-Smad2, Smad2 and phospho-Smad3 were measured by Western Blot. B. The bands were semi-quantitatively analyzed by using Image J software, normalized to β-actin density. Data represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. NC group. ##P < 0.01 vs. HG + mimics group.